The invention relates to a method and apparatus for mixing a thin film of fluid. More particularly, the invention relates to a method and apparatus for mixing a thin film of chemical, biochemical, or biological fluid undergoing a reaction such as occurs in a chemical or biological assay. Even more particularly, the invention relates to a method and apparatus for mixing a thin film of fluid undergoing a surface reaction such as occurs in a chemical or biological assay.
In many thin film reactions, for example, thin film reactions involving chemical, biochemical, or biological fluids, there is inadequate mixing at the interface between the fluids or between the fluid and the reactive surface. inadequate mixing may occur, for example, because the interface or surface area for reaction is very large and the available fluid sample volume is very small, resulting in a thin film of fluid being spread across the reactive interface or surface which may be on the order of a few microns to a few millimeters thick. In such situations, the fluid may not adequately contact the entire reactive interface or surface. The surface tension or density of the fluid involved in the reaction may be such that adequate mixing of the fluid is difficult to obtain, resulting in inhomogeneities in composition and inconsistent distribution of reactant fluid across the reactive interface or surface. Furthermore, the components of interest in the fluid, such as the components actually involved in the reaction, are frequently present in very low concentrations. Inadequate mixing therefore can cause significant boundary layers or lead to diffusion limited reactions which deleteriously affect the sensitivity or specificity of the reaction, rate of reaction, extent of reaction, homogeneity, or percentage yield.
Inadequate mixing is a particular problem in chemical and biological assays in which very small samples of chemical, biochemical, or biological fluids are typically reacted. For example, the ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary to reveal the changes in genes which confer disease. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides.
One typical method involves hybridization with either target or probe nucleotide sequences immobilized onto a solid support. The stationary phase consists of either numerous targets or probes immobilized onto a solid support having a surface area of typically less than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of either the targets or probes. The mobile phase containing either the probes or targets is spread or placed on the support, which is then covered with a slide of glass or fused silica. The covered slide is then preferably placed in a environmentally controlled chamber such as an incubator and the hybridization reaction is allowed to proceed. The reactants (either targets or probes) in the mobile phase diffuse through the mobile phase to the interface or surface where the complementary probes or targets are immobilized. Preferably, the mobile phase reactants are labeled, such as through the use of fluorescent tags, so that the identities of the targets and probes undergoing hybridization can be identified and monitored. Alternatively, a detectable label is attached to the hybridized target-probe pair after the hybridization reaction is completed. The hybridization reaction typically takes place over a few minutes to many hours.
DNA arrays have recently been developed for such hybridization reactions in which, for example, photolithographic or deposition methods are used to construct the arrays of oligonucleotides and fluorescence scanning is used to detect the molecular binding events which occur at different points in the array. For descriptions of DNA arrays and associated techniques, see, for example, Kreiner, "Rapid Genetic Sequence Analysis Using A DNA Probe Array System," American Laboratory, March, 1996, pp. 39-43; Lipshutz et al., "Using Oligonucleotide Probe Arrays To Access Genetic Diversity," BioTechniques, Vol. 19, No. 3, 1995, pp. 442-447; Fodor et al., "Light-Directed, Spatially Addressable Parallel Chemical Synthesis," Science, Vol. 251, Feb. 15, 1991, pp. 767-773; Medlin, "The Amazing Shrinking Laboratory," Environmental Health Perspectives, Vol. 103, No. 3, March, 1995, pp. 244-246; Southern et al., "Analyzing And Comparing Nucleic Acid Sequences By Hybridization To Arrays Of Oligonucleotides: Evaluation Using Experimental Models," Genomics, Vol. 13, 1992, pp. 1008-1017; and Gacia et al., "Detection Of Heterozygous Mutations In BRCA1 Using High Density Oligonucleotide Arrays And Two-Colour Fluorescence Analysis," Nature Genetics, Vol. 14, December, 1996, pp. 441-447; all incorporated herein by reference. See also, U.S. Pat. Nos. 5,585,639; 5,601,980; and 5,551,487; all incorporated herein by reference.
Problems are frequently encountered in conducting chemical or biological assays, including use of arrays, with poor hybridization kinetics and efficiency or reaction specificity and sensitivity, since diffusion generally is the only means of moving the reactants in the mobile phase to the interface or surface containing the immobilized reactants. Alternatively, the fluid sample must be removed from the reaction chamber, mixed in separate chambers external to the reaction chamber, and then reintroduced into the reaction chamber. Valuable fluid sample is wasted or lost in the separate external chambers required in such mixing process.
What is needed is a method and apparatus for mixing a thin film of fluid, particularly a chemical, biochemical, or biological fluid undergoing a reaction, which induce sufficient mixing to overcome boundary layer effects and diffusion limited reaction rates. What is further needed is a method and apparatus which is convenient and easy to use, which may be adapted for processing large numbers of samples, which is reproducible and controllable, which minimizes the required sample volume, and which is adapted to automated use.